Polypeptide-polymer conjugates and methods of use thereof

ABSTRACT

The present invention provides polypeptide-polymer conjugates. A subject polypeptide-polymer conjugate is useful in a variety of applications, which are also provided.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/900,011, filed Feb. 20, 2018, now U.S. Pat. No. 10,350,267, which isa continuation of U.S. patent application Ser. No. 15/214,398, filedJul. 19, 2016, now U.S. Pat. No. 9,925,237, which is a continuation ofU.S. patent application Ser. No. 12/933,655, filed Nov. 10, 2010, nowU.S. Pat. No. 9,428,561, which is a national stage filing under 35U.S.C. § 371 of PCT/US2009/038446, filed Mar. 26, 2009, which claims thebenefit of U.S. Provisional Patent Application No. 61/040,556, filedMar. 28, 2008, which applications are incorporated herein by referencein their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. AR047304awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

The use of chemical tethers to create solid-phase forms of biologicallyactive agents is a recurring theme across a wide range of medical andbiological applications. Chemical tethers can be used to attachbioactive peptides or proteins to surfaces, to impart bioactivity toporous or hydrogel implants, or in drug delivery applications.Solid-phase presentation can alter the way that bioactive moleculesfunction in a biological setting.

SUMMARY OF THE INVENTION

The present invention provides polypeptide-polymer conjugates. A subjectpolypeptide-polymer conjugate is useful in a variety of applications,which are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a bioconjugate scheme to graft a recombinant protein(Shh, sonic hedgehog) to the polymer hyaluronic acid (HyA).

FIGS. 2A and 2B depict gel electrophoresis of Shh and its conjugationproducts with poly(acrylic acid) (pAAc) (FIG. 2A) and with HyA (FIG.2B).

FIG. 3 depicts a schematic of Shh signal transduction pathway and aproposed mechanism for impact of multivalency of Shh on its bioactivity.

FIG. 4 depicts C3H10T1/2 bioactivity results against soluble Shh (♦,heavy line), soluble Shh with soluble HyA (▪, dashed lines), and theShh-HyA conjugates in stoichiometric ratios of 0.6:1 (Δ), 3.5:1 (◯), 7:1(⋄), 14:1 (□), and 22:1 (x).

FIGS. 5A-C present a panel of photomicrographs depicting chickchorioallantoic membrane (CAM) reactions to negative control samples(FIG. 5A), freely soluble Shh (FIG. 5B), and the 14:1 Shh/HyAmultivalent form (FIG. 5C).

FIG. 6 depicts quantitative results of angiogenesis in the CAM assayderived from photomicrograph image analysis.

FIG. 7 depicts numerical model results of Shh-HyA conjugate bioactivityin C3H10T1/2 cells. The upper panel presents activity as a function ofShh concentration for the stoichiometric ratios 1:1-30:1 for a modelincorporating steric interaction. The lower panel presents a plot ofEC₅₀ versus substitution level for two types of models versusexperimental results.

DEFINITIONS

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term “polypeptide”includes fusion proteins, including, but not limited to, fusion proteinswith a heterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like. The term“polypeptide” includes polypeptides comprising one or more of a fattyacid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety.The term “polypeptides” includes post-translationally modifiedpolypeptides.

As used herein, the term “copolymer” describes a polymer which containsmore than one type of subunit. The term encompasses polymer whichinclude two, three, four, five, or six types of subunits.

The terms “subject,” “individual,” “host,” and “patient” are usedinterchangeably herein to a member or members of any mammalian ornon-mammalian species. Subjects and patients thus include, withoutlimitation, humans, non-human primates, canines, felines, ungulates(e.g., equine, bovine, swine (e.g., pig)), avians, rodents (e.g., rats,mice), and other subjects. Non-human animal models, particularlymammals, e.g. a non-human primate, a murine (e.g., a mouse, a rat),lagomorpha, etc. may be used for experimental investigations.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the condition, i.e., causing aclinical symptom to not significantly develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its symptoms, or(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, is sufficient, in combination withanother agent, or alone in one or more doses, to effect such treatmentfor the disease. The “therapeutically effective amount” will varydepending on the compound, the disease and its severity and the age,weight, etc., of the subject to be treated.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” and “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and adjuvantthat are useful in preparing a pharmaceutical composition that aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use as well as human pharmaceuticaluse. “A pharmaceutically acceptable excipient, diluent, carrier andadjuvant” as used in the specification and claims includes one and morethan one such excipient, diluent, carrier, and adjuvant.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asynthetic substrate” includes a plurality of such substrates andreference to “the recombinant polypeptide” includes reference to one ormore recombinant polypeptides and equivalents thereof known to thoseskilled in the art, and so forth. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present invention provides polypeptide-polymer conjugates, wheresuch conjugates have controlled attachment stoichiometry. A subjectpolypeptide-polymer conjugate is useful in a variety of applications,which are also provided.

In some embodiments, a subject polypeptide-polymer conjugate is of theformula:

X—(Y)_(n)—Z,

where X is a biologically active polypeptide;

Y is an optional linker moiety, such that n is 0 or an integer from 1 toabout 10; and

Z is a biocompatible polymer comprising from about 50 to 100,000subunits.

The biological activity of a polypeptide conjugated to the polymersubstrate is enhanced relative to the activity of the polypeptide insoluble form, e.g., compared to the activity of the polypeptide notconjugated to the polymer. In some embodiments, the biological activityof the polypeptide of a subject polypeptide-polymer conjugate is atleast about 25%, at least about 50%, at least about 75%, at least about2-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 20-fold, at least about 25-fold, at least about30-fold, at least about 40-fold, at least about 50-fold, at least about75-fold, at least about 100-fold, at least about 200-fold, at leastabout 500-fold, or at least about 1000-fold, or more than 1000-fold,greater than the biological activity of the polypeptide in soluble(unconjugated) form.

In some embodiments, the biological activity of the polypeptide of asubject polypeptide-polymer conjugate is at least about 25%, at leastabout 50%, at least about 75%, at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, at least about 30-fold, at least about40-fold, at least about 50-fold, at least about 75-fold, at least about100-fold, at least about 200-fold, at least about 500-fold, or at leastabout 1000-fold, or more than 1000-fold, greater than the biologicalactivity of the polypeptide in when conjugated to the polymer at a 1:1molar ratio.

In some embodiments, the biological activity of the polypeptide of asubject polypeptide-polymer conjugate is at least about 25%, at leastabout 50%, at least about 75%, at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, at least about 30-fold, at least about40-fold, at least about 50-fold, at least about 75-fold, at least about100-fold, at least about 200-fold, at least about 500-fold, or at leastabout 1000-fold, or more than 1000-fold, greater than the biologicalactivity of the polypeptide when present in admixture with the polymer.

For example, in some embodiments, the EC₅₀ of the polypeptide of asubject polypeptide-polymer conjugate is at least about 25%, at leastabout 50%, at least about 75%, at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, at least about 30-fold, at least about40-fold, at least about 50-fold, at least about 75-fold, at least about100-fold, at least about 200-fold, at least about 500-fold, or at leastabout 1000-fold, or more than 1000-fold, lower than the EC₅₀ of thepolypeptide in soluble (unconjugated form).

Whether the biological activity of the polypeptide of a subjectpolypeptide-polymer conjugate is increased relative to the biologicalactivity of the polypeptide in soluble (unconjugated) form is readilydetermined using an appropriate assay(s) for the biological activity.

The molar ratio of the polypeptide to the polymer can vary from about5:1 to about 100:1, e.g., from about 5:1 to about 7:1, from about 7:1 toabout 10:1, from about 10:1 to about 12:1, from about 12:1 to about15:1, from about 15:1 to about 20:1, from about 20:1 to about 25:1, fromabout 25:1 to about 30:1, from about 30:1 to about 35:1, from about 35:1to about 40:1, from about 40:1 to about 45:1, from about 45:1 to about50:1, from about 50:1 to about 60:1, from about 60:1 to about 70:1, fromabout 70:1 to about 80:1, from about 80:1 to about 90:1, or from about90:1 to about 100:1.

For example, where a subject polypeptide polymer conjugate comprises apolypeptide that induces angiogenesis (e.g., the polypeptide is anangiogenic polypeptide), in some embodiments, the angiogenic polypeptideof a subject polypeptide-polymer conjugate induces at least about 25%,at least about 50%, at least about 75%, at least about 2-fold, at leastabout 5-fold, at least about 10-fold, at least about 15-fold, at leastabout 20-fold, at least about 25-fold, at least about 30-fold, at leastabout 40-fold, at least about 50-fold, at least about 75-fold, at leastabout 100-fold, at least about 200-fold, at least about 500-fold, or atleast about 1000-fold, or more than 1000-fold, more angiogenesis thanthe angiogenic polypeptide when present in admixture with the polymer,when in soluble (unconjugated) form, or when conjugated to the polymerat a 1:1 molar ratio.

Polymers

Suitable polymers to which a biologically active polypeptide isconjugated include biocompatible polymers comprising from about 50 toabout 100,000 subunits, e.g., from about 50 subunits to about 100subunits, from about 100 subunits to about 500 subunits, from about 500subunits to about 1,000 subunits, from about 1,000 subunits to about5,000 subunits, from about 5,000 subunits to about 10,000 subunits, fromabout 10,000 subunits to about 25,000 subunits, from about 25,000subunits to about 50,000 subunits, or from about 50,000 subunits toabout 100,000 subunits. In some embodiments, the linear polymercomprises more than 100,000 subunits.

The subunits can all be identical, e.g., the polymer is a homopolymer.In other embodiments, more than one species of subunit is present, e.g.,the polymer is a heteropolymer or co-polymer. In some embodiments, thepolymer is a linear polymer. In other embodiments, the polymer mayinclude one or more branches.

Suitable polymers include natural polymers, semisynthetic polymers, andsynthetic polymers.

Suitable natural polymers include hyaluronic acid, collagen,glycosaminoglycans, cellulose, polysaccharides, and the like.

Suitable semisynthetic polymers include, but are not limited to,collagen crosslinked with aldehydes or precursors of the same,dicarboxylic acids or their halogenides, diamines, derivatives ofcellulose, hyaluronic acid, chitin, chitosan, gellan gum, xanthan,pectin or pectic acid, polyglycans, polymannan, agar, agarose, naturalgums and glycosaminoglycans.

Suitable synthetic polymers include, but are not limited to, polymers orcopolymers derived from polydioxane, polyphosphazene, polysulphoneresins, poly(acrylic acid), poly(acrylic acid) butyl ester,poly(ethylene glycol), poly(propylene), polyurethane resins,poly(methacrylic acid), poly(methacrylic acid)-methyl ester,poly(methacrylic acid)-n butyl ester, poly(methacrylic acid)-t butylester, polytetrafluoroethylene, polyperfluoropropylene, poly N-vinylcarbazole, poly(methyl isopropenyl ketone), poly alphamethyl styrene,polyvinylacetate, poly(oxymethylene), poly(ethylene-co-vinyl acetate), apolyurethane, a poly(vinyl alcohol), and polyethylene terephthalate;ethylene vinyl alcohol copolymer (commonly known by the generic nameEVOH or by the trade name EVAL); polybutylmethacrylate;poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone;poly(lactide-co-glycolide); poly(hydroxybutyrate);poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester;polyanhydride; poly(glycolic acid) (PGA); poly(D,L-lactic acid) (PLA);copolymers of PGA and PLA; poly(glycolic acid-co-trimethylenecarbonate); polyphosphoester; polyphosphoester urethane; poly(aminoacids); cyanoacrylates; poly(trimethylene carbonate);poly(iminocarbonate); copoly(ether-esters) (e.g., PEO/PLA); polyalkyleneoxalates; polyphosphazenes; polyurethanes; silicones; polyesters;polyolefins; polyisobutylene and ethylene-alphaolefin copolymers;acrylic polymers and copolymers; vinyl halide polymers and copolymers,such as polyvinyl chloride; polyvinyl ethers, such as polyvinyl methylether; polyvinylidene halides, such as polyvinylidene fluoride andpolyvinylidene chloride; polyacrylonitrile; polyvinyl ketones; polyvinylaromatics, such as polystyrene; polyvinyl esters, such as polyvinylacetate; copolymers of vinyl monomers with each other and olefins, suchas ethylene-methyl methacrylate copolymers, acrylonitrile-styrenecopolymers, ABS resins, and ethylene-vinyl acetate copolymers;polyamides, such as Nylon 66 and polycaprolactam; alkyd resins;polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins;polyurethanes; rayon; rayon-triacetate; cellulose; cellulose acetate;cellulose butyrate; cellulose acetate butyrate; cellophane; cellulosenitrate; cellulose propionate; cellulose ethers; amorphous Teflon; andcarboxymethyl cellulose.

The polymer to which the biologically active polypeptide is conjugatedcan comprise multiple subunits selected from hyaluronic acid, acrylicacid, ethylene glycol, vinyl, propylene, methyl methacrylate,methacrylic acid, acrylamide, hydroxyethyl methacrylate,tetrafluoroethylene, oxymethylene, a sugar (e.g., glucose, mannitol,maltose, arabinose, etc.), taurine, betaine, modified celluloses,hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl methyl cellulose, carboxymethylcellulose, modified starches, hydrophobically modified starch,hydroxyethyl starch, hydroxypropyl starch, amylose, amylopectin,oxidized starch, an amino acid, and copolymers of any of the foregoing.In some embodiments, the polymer does not include amino acids.

In some embodiments, the polymer is hyaluronic acid or a hyaluronic acidderivative. Hyaluronic acid derivatives include, e.g., a hyaluronic acidester where part or all of the carboxylic functions are esterified withan alcohol of the aliphatic, aromatic, arylaliphatic, cycloaliphatic orheterocyclic series; a hemiester of succinic acid or a heavy metal saltof the hemiester of succinic acid with hyaluronic acid or with a partialor total ester of hyaluronic acid; sulphated or N-sulphated hyaluronicacid;

Polypeptides

The polypeptide component of a subject polypeptide-polymer conjugate isbiologically active, e.g., exhibits one or more biological activities invivo and/or in vitro. Biological activities include, e.g., antigenbinding; activation of a signaling pathway in a eukaryotic cell;induction of cell proliferation; induction of cell differentiation;induction of angiogenesis; induction of apoptosis; induction ofangiogenesis; inhibition of angiogenesis; reduction of coagulation;reduction of cell adhesion; enhancement of cell adhesion; control ofcell fate; and the like.

The polypeptide component of a subject polypeptide-polymer conjugate canbe a naturally-occurring polypeptide, a recombinant polypeptide, or asynthetic polypeptide. The polypeptide can comprise one or morenon-amino acid moieties, e.g., a lipid moiety, a sugar moiety, acarbohydrate moiety, etc.

In some embodiments, a single species of polypeptide is attached to apolymer, e.g., a plurality of polypeptides, all having the same aminoacid sequence, is attached to a polymer. In other embodiments, two ormore species of polypeptides are attached to a polymer, where a firstpolypeptide has a first amino acid sequence, and a second polypeptidehas a second amino acid sequence that is different from the first aminoacid sequence (e.g., where the second amino acid sequence has from about95% to about 99%, from about 90% to about 95%, from about 85% t about90%, from about 80% to about 85%, from about 75% to about 80%, fromabout 70% to about 75%, from about 65% to about 70%, or less than 65%,amino acid sequence identity with the first amino acid sequence). Forexample, the first and the second polypeptides could target differentcell surface receptors, e.g., the first polypeptide could provide forcell adhesion through an integrin receptor, and the second polypeptidecould provide for activation of a bound cell, e.g., via growth factorreceptors, etc. As another example, the first and the secondpolypeptides could induce cell differentiation, e.g., the first and thesecond polypeptides could both induce myogenesis, the first and thesecond polypeptides could both induce cardiomyogenesis, the first andthe second polypeptides could both induce neurogenesis, the first andthe second polypeptides could both induce differentiation of aprogenitor cell into a chondrocyte, or the first and the secondpolypeptides could both induce hematopoiesis, in a target totipotent,pluripotent, or multipotent progenitor cell.

In some embodiments, the polypeptide component of a subjectpolypeptide-polymer conjugate is recombinant, e.g., the polypeptideincludes one or more amino acids that are not normally in amide bondlinkage with the polypeptide. For example, the polypeptide can beengineered to include an amino acid that facilitates linkage to thepolymer component of the polypeptide-polymer conjugate. As an example,the polypeptide can be engineered to include a cysteine residue thatfacilitates linkage to the polymer component of the polypeptide-polymerconjugate.

The size of the polypeptide can range from 2 kDa to about 2000 kDa,e.g., from about 2 kDa to about 5 kDa, from about 5 kDa to about 10 kDa,from about 10 kDa to about 25 kDa, from about 25 kDa to about 50 kDa,from about 50 kDa to about 100 kDa, from about 100 kDa to about 250 kDa,from about 250 kDa to about 500 kDa, from about 500 kDa to about 1000kDa, from about 1000 kDa to about 2000 kDa.

In some embodiments, the polypeptide component of a subjectpolypeptide-polymer conjugate comprises a detectable label. Suitablelabels include, e.g., radioisotopes (e.g., ¹²⁵I; ³⁵S, and the like);enzymes whose products generate a detectable signal (e.g., luciferase,β-galactosidase, horse radish peroxidase, alkaline phosphatase, and thelike); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu,or others of the lanthanide series, attached to the antibody throughmetal chelating groups such as EDTA; chemiluminescent compounds, e.g.,luminol, isoluminol, acridinium salts, and the like; bioluminescentcompounds, e.g., luciferin; fluorescent proteins (e.g., a greenfluorescent protein, a yellow fluorescent protein, a red fluorescentprotein, etc.); and the like.

Polypeptides that are of interest for attachment to a polymer, togenerate a subject polypeptide-polymer conjugate include, e.g., growthfactors, receptors, polypeptide ligands for receptors, enzymes,antibodies, coagulation factors, anti-coagulation factors, angiogenicfactors, anti-angiogenic factors, etc. Suitable polypeptides includelinear polypeptides and cyclic polypeptides. Suitable polypeptidesinclude naturally occurring polypeptides, synthetic polypeptides, andthe like.

Suitable polypeptides include, but are not limited to, an interferon(e.g., IFN-γ, IFN-α, IFN-β, IFN-ω; IFN-τ); an insulin (e.g., Novolin,Humulin, Humalog, Lantus, Ultralente, etc.); an erythropoietin (“EPO”;e.g., Procrit®, Eprex®, or Epogen® (epoetin-α); Aranesp®(darbepoietin-α); NeoRecormon®, Epogin® (epoetin-β); and the like); anantibody (e.g., a monoclonal antibody) (e.g., Rituxan® (rituximab);Remicade® (infliximab); Herceptin® (trastuzumab); Humira™ (adalimumab);Xolair® (omalizumab); Bexxar® (tositumomab); Raptiva™ (efalizumab);Erbitux™ (cetuximab); and the like), including an antigen-bindingfragment of a monoclonal antibody; a blood factor (e.g., Activase®(alteplase) tissue plasminogen activator; NovoSeven® (recombinant humanfactor VIIa); Factor VIIa; Factor VIII (e.g., Kogenate®); Factor IX;β-globin; hemoglobin; and the like); a colony stimulating factor (e.g.,Neupogen® (filgrastim; G-CSF); Neulasta (pegfilgrastim); granulocytecolony stimulating factor (G-CSF), granulocyte-monocyte colonystimulating factor, macrophage colony stimulating factor, megakaryocytecolony stimulating factor; and the like); a growth hormone (e.g., asomatotropin, e.g., Genotropin®, Nutropin®, Norditropin®, Saizen®,Serostim®, Humatrope®, etc.; a human growth hormone; and the like); aninterleukin (e.g., IL-1; IL-2, including, e.g., Proleukin®; IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9; etc.); a growth factor (e.g., Regranex®(beclapermin; PDGF); Fiblast® (trafermin; bFGF); Stemgen® (ancestim;stem cell factor); keratinocyte growth factor; an acidic fibroblastgrowth factor, a stem cell factor, a basic fibroblast growth factor, ahepatocyte growth factor; and the like); a receptor (e.g., aTNF-α-binding soluble receptor such as Enbrel® (etanercept); a VEGFreceptor; a interleukin receptor; a γ/δ T cell receptor; and the like);a neurotransmitter receptor (e.g., a nicotinic acetylcholine receptor, aglutamate receptor, a GABA receptor, etc.); an enzyme (e.g.,α-glucosidase; Cerazyme® (imiglucarase; β-glucocerebrosidase, Ceredase®(alglucerase;); an enzyme activator (e.g., tissue plasminogenactivator); a chemokine (e.g., IP-10; Mig; Groα/IL-8, RANTES; MIP-1α;MIP-1β; MCP-1; PF-4; and the like); an angiogenic agent (e.g., vascularendothelial growth factor (VEGF); an anti-angiogenic agent (e.g., a VEGFreceptor); a neuroactive peptide such as bradykinin, cholecystokinin,gastin, secretin, oxytocin, gonadotropin-releasing hormone,beta-endorphin, enkephalin, substance P, somatostatin, prolactin,galanin, growth hormone-releasing hormone, bombesin, dynorphin,neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone,calcitonin, insulin, glucagon, vasopressin, angiotensin II,thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleeppeptide, etc.; other proteins such as a thrombolytic agent, an atrialnatriuretic peptide, bone morphogenic protein, thrombopoietin, relaxin,glial fibrillary acidic protein, follicle stimulating hormone, a humanalpha-1 antitrypsin, a leukemia inhibitory factor, a transforming growthfactor, an insulin-like growth factor, a luteinizing hormone, amacrophage activating factor, tumor necrosis factor, a neutrophilchemotactic factor, a nerve growth factor a tissue inhibitor ofmetalloproteinases; a vasoactive intestinal peptide, angiogenin,angiotropin, fibrin; hirudin; a leukemia inhibitory factor; an IL-1receptor antagonist (e.g., Kineret® (anakinra)); an ion channel, e.g.,cystic fibrosis transmembrane conductance regulator (CFTR); dystrophin;utrophin, a tumor suppressor; lysosomal enzyme acid α-glucosidase (GAA);and the like.

Suitable polypeptides include sonic hedgehog (Shh), bone morphogenicprotein-4, interleukin-3 (IL-3), stem cell factor-1 (SCF-1), fms-liketyrosine kinase-3 (Flt3) ligand, leukemia inhibitory factor (LIF),fibroblast growth factor-2 (FGF-2), and epidermal growth factor (EGF).Suitable polypeptides include brain-derived neurotrophic factor (BDNF),nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4),neurotrophin-5 (NT-5), basic fibroblast growth factor (bFGF),insulin-like growth factor-1 (IGF-1), glial-derived neurotrophic factor(GDNF), and protease nexin-1. Suitable angiogenic polypeptides include anetrin-1 polypeptide, a vascular endothelial growth factor (VEGF)polypeptide, a platelet-derived growth factor (PDGF) polypeptide, afibroblast growth factor (FGF) polypeptide, and an angiopoietinpolypeptide.

Suitable polypeptides also include clotting factors, e.g., thrombin,etc. Suitable polypeptides also include anti-coagulants. Suitablepolypeptides also include cell-binding polypeptides.

Suitable polypeptides also include, e.g., Nestin, Vimentin,Prominin/CD133, Sonic hedgehog and other hedgehog ligands, Wnt ligands,Neurocan/tenascin C, Nun 1, Pax-6, Sox-2, Musashi-1, NG2/CSPG-4, NeuroD3, Neurogenin 1, and active fragments and subsequences of any thesepolypeptides.

Suitable polypeptides also include, e.g., β tubulin III, MAP2, Neuronspecific enolase, NCAM, CD24, HAS, Synapsin I, Synaptophysin, CAMK Iia,Tyrosine hydroxylase, Glutamate transporter, Glutamate receptor, Cholinereceptor, nicotinic A2, EphB2, GABA-A receptor, Serotonin (5HT-3)receptor, Choline acetyltransferase, and fragments and subsequences ofany of the foregoing.

Suitable polypeptides also include, e.g., a calcium channel; a T-cellantigen receptor; a chemokine receptor; a potassium channel; aneurotransmitter receptor (e.g., a serotonin receptor; a GABA receptor;a glutamate receptor; a nicotinic acetylcholine receptor; etc.); agrowth factor receptor (e.g., epidermal growth factor receptor; vascularendothelial growth factor receptor, etc.); a bone morphogenetic protein;a polypeptide that activates a cell signaling pathway; and the like.

The polypeptide component of a subject polypeptide-polymer conjugate isbiologically active. Those skilled in the art can readily determinewhether a given polypeptide is biologically active, using any of anumber of well-known assays designed to test for a particular biologicalactivity. Examples of useful assays for particular biologically activepolypeptides include, but are not limited to, GMCSF (Eaves, A. C. andEaves C. J., Erythropoiesis in culture. In: McCullock E A (edt) Cellculture techniques—Clinics in hematology. W B Saunders, Eastbourne, pp371-91 (1984); Metcalf, D., International Journal of Cell Cloning 10:116-25 (1992); Testa, N. G., et al., Assays for hematopoietic growthfactors. In: Balkwill F R (edt) Cytokines A practical Approach, pp229-44; IRL Press Oxford 1991) EPO (bioassay: Kitamura et al., J. Cell.Physiol. 140 p323 (1989)); Hirudin (platelet aggregation assay: BloodCoagul Fibrinolysis 7(2):259-61 (1996)); IFNα (anti-viral assay:Rubinstein et al., J. Virol. 37(2):755-8 (1981); anti-proliferativeassay: Gao Y, et al Mol Cell Biol. 19(11):7305-13 (1999); and bioassay:Czarniecki et al., J. Virol. 49 p490 (1984)); GCSF (bioassay: Shirafujiet al., Exp. Hematol. 17 p116 (1989); proliferation of murine NFS-60cells (Weinstein et al, Proc Natl Acad Sci 83:5010-4 (1986)); insulin(³H-glucose uptake assay: Steppan et al., Nature 409(6818):307-12(2001)); hGH (Ba/F3-hGHR proliferation assay: J Clin Endocrinol Metab85(11):4274-9 (2000); International standard for growth hormone: HormRes, 51 Suppl 1:7-12 (1999)); factor X (factor X activity assay: VanWijk et al. Thromb Res 22:681-686 (1981)); factor VII (coagulation assayusing prothrombin clotting time: Belaaouaj et al., J. Biol. Chem.275:27123-8(2000); Diaz-Collier et al., Thromb Haemost 71:339-46(1994)).

Assays for activation of a cell signaling pathway are known in the art.Assays for induction of cell proliferation are known in the art, andinclude, e.g., ³H-thymidine uptake assays, etc. Assays for induction ofangiogenesis include, e.g., a chick chorioallantoic membrane (CAM)assay, an in vitro endothelial cell assay, a Matrigel assay, a discangiogenesis system, and the like. Assays for induction of celldifferentiation are known in the art, and include assays to detect geneproduct(s) associated with a differentiated cell type.

Linkers

As noted above, in some embodiments, a subject polypeptide-polymerconjugate comprises a linker group that links the polypeptide to thepolymer. Suitable linkers include peptide linkers, and non-peptidelinkers.

A linker peptide may have any of a variety of amino acid sequences.Exemplary peptide linkers are between about 6 and about 40 amino acidsin length, or between about 6 and about 25 amino acids in length.Exemplary linkers include poly(glycine) linkers (e.g., (Gly)_(n), wheren is an integer from 2 to about 10); linkers comprising Gly and Ser; andthe like.

Conjugation

A variety of conjugation methods and chemistries can be used toconjugate a polypeptide to a polymer. Various zero-length,homo-bifunctional, and hetero-bifunctional crosslinking reagents can beused. Zero-length crosslinking reagents include direct conjugation oftwo intrinsic chemical groups with no introduction of extrinsicmaterial. Agents that catalyze formation of a disulfide bond belong tothis category. Another example is reagents that induce condensation of acarboxyl and a primary amino group to form an amide bond such ascarbodiimides, ethylchloroformate, Woodward's reagent K(2-ethyl-5-phenylisoxazolium-3′-sulfonate), and carbonyldiimidazole.Homo- and hetero-bifunctional reagents generally contain two identicalor two non-identical sites, respectively, which may be reactive withamino, sulfhydryl, guanidino, indole, or nonspecific groups.

In some embodiments, the polymer comprises an amino-reactive group forreacting with a primary amine group on the polypeptide, or on a linker.Suitable amino-reactive groups include, but are not limited to,N-hydroxysuccinimide (NHS) esters, imidoesters, isocyanates,acylhalides, arylazides, p-nitrophenyl esters, aldehydes, and sulfonylchlorides.

In some embodiments, the polymer comprises a sulfhydryl-reactive group,e.g., for reacting with a cysteine residue in the polypeptide. Suitablesulfhydryl-reactive groups include, but are not limited to, maleimides,alkyl halides, pyridyl disulfides, and thiophthalimides.

In other embodiments, carbodiimides soluble in both water and organicsolvent, are used as carboxyl-reactive reagents. These compounds reactwith free carboxyl groups forming a pseudourea that can then couple toavailable amines, yielding an amide linkage.

As noted above, in some embodiments, a polypeptide is conjugated to apolymer using a homobifunctional crosslinker.

In some embodiments, the homobifunctional crosslinker is reactive withprimary amines. Homobifunctional crosslinkers that are reactive withprimary amines include NHS esters, imidoesters, isothiocyanates,isocyanates, acylhalides, arylazides, p-nitrophenyl esters, aldehydes,and sulfonyl chlorides.

Non-limiting examples of homobifunctional NHS esters includedisuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartarate (DST),disulfosuccinimidyl tartarate (sulfo-DST),bis-2-(succinimidooxycarbonyloxy)ethylsulfone (BSOCOES),bis-2-(sulfosuccinimidooxycarbonyloxy)ethylsulfone (sulfo-BSOCOES),ethylene glycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS),dithiobis(succinimidylpropionate (DSP), anddithiobis(sulfosuccinimidylpropionate(sulfo-DSP). Non-limiting examplesof homobifunctional imidoesters include dimethyl malonimidate (DMM),dimethyl succinimidate (DMSC), dimethyl adipimidate (DMA), dimethylpimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-oxydipropionimidate (DODP),dimethyl-3,3′-(methylenedioxy)dipropionimidate (DMDP),dimethyl-,3′-(dimethylenedioxy)dipropionimidate (DDDP),dimethyl-3,3′-(tetramethylenedioxy)dipropionimidate (DTDP), anddimethyl-3,3′-dithiobispropionimidate (DTBP).

Non-limiting examples of homobifunctional isothiocyanates include:p-phenylenediisothiocyanate (DITC), and4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). Non-limitingexamples of homobifunctional isocyanates include xylene-diisocyanate,toluene-2,4-diisocyanate, toluene-2-isocyanate-4-isothiocyanate,3-methoxydiphenylmethane-4,4′-diisocyanate,2,2′-dicarboxy-4,4′-azophenyldiisocyanate, andhexamethylenediisocyanate. Non-limiting examples of homobifunctionalacylhalides include 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and4,4′-difluoro-3,3′-dinitrophenyl-sulfone. Non-limiting examples ofhomobifunctional aliphatic aldehyde reagents include glyoxal,malondialdehyde, and glutaraldehyde. Non-limiting examples ofhomobifunctional acylating reagents include nitrophenyl esters ofdicarboxylic acids. Non-limiting examples of homobifunctional aromaticsulfonyl chlorides include phenol-2,4-disulfonyl chloride, andα-naphthol-2,4-disulfonyl chloride. Non-limiting examples of additionalamino-reactive homobifunctional reagents include erythritolbiscarbonate,which reacts with amines to give biscarbamates.

In some embodiments, the homobifunctional crosslinker is reactive withfree sulfhydryl groups. Homobifunctional crosslinkers reactive with freesulfhydryl groups include, e.g., maleimides, pyridyl disulfides, andalkyl halides.

Non-limiting examples of homobifunctional maleimides includebismaleimidohexane (BMH), N,N′-(1,3-phenylene) bismaleimide,N,N′-(1,2-phenylene)bismaleimide, azophenyldimaleimide, andbis(N-maleimidomethyl)ether. Non-limiting examples of homobifunctionalpyridyl disulfides include1,4-di-3′-(2′-pyridyldithio)propionamidobutane (DPDPB). Non-limitingexamples of homobifunctional alkyl halides include2,2′-dicarboxy-4,4′-diiodoacetamidoazobenzene, a,a′-diiodo-p-xylenesulfonic acid, a, a′-dibromo-p-xylenesulfonic acid,N,N′-bis(b-bromoethyl)benzylamine, N,N′-di(bromoacetyl)phenylhydrazine,and 1,2-di(bromoacetyl)amino-3-phenylpropane.

As noted above, in some embodiments, a polypeptide is conjugated to apolymer using a heterobifunctional reagent. Suitable heterobifunctionalreagents include amino-reactive reagents comprising a pyridyl disulfidemoiety; amino-reactive reagents comprising a maleimide moiety;amino-reactive reagents comprising an alkyl halide moiety; andamino-reactive reagents comprising an alkyl dihalide moiety.

Non-limiting examples of hetero-bifunctional reagents with a pyridyldisulfide moiety and an amino-reactive NHS ester includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (LC-SPDP), sulfosuccinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (sulfo-LCSPDP),4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT),and sulfosuccinimidyl 6-α-methyl-α-(2-pyridyldithio)toluamidohexanoate(sulfo-LC-SMPT).

Non-limiting examples of heterobifunctional reagents comprising amaleimide moiety and an amino-reactive NHS ester include succinimidylmaleimidylacetate (AMAS), succinimidyl 3-maleimidylpropionate (BMPS),N-.gamma.-maleimidobutyryloxysuccinimide ester(GMBS)N-.gamma.-maleimidobutyryloxysulfosuccinimide ester (sulfo-GMBS)succinimidyl 6-maleimidylhexanoate (EMCS), succinimidyl3-maleimidylbenzoate (SMB), m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester(sulfo-MBS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

Non-limiting examples of heterobifunctional reagents comprising an alkylhalide moiety and an amino-reactive NHS ester includeN-succinimidyl-(4-iodoacetyl)aminobenzoate (SIAB),sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate (sulfo-SIAB),succinimidyl-6-(iodoacetyl)aminohexanoate (SIAX),succinimidyl-6-(6-((iodoacetyl)-amino)hexanoylamino)hexanoate (SIAXX),succinimidyl-6-(((4-(iodoacetyl)-amino)methyl)-cyclohexane-1-carbonyliaminohexanoate(SIACX), andsuccinimidyl-4((iodoacetyl)-amino)methylcyclohexane-1-carboxylate(SIAC).

A non-limiting example of a hetero-bifunctional reagent comprising anamino-reactive NHS ester and an alkyl dihalide moiety isN-hydroxysuccinimidyl 2,3-dibromopropionate (SDBP). A non-limitingexample of a hetero-bifunctional reagent comprising an alkyl halidemoiety and an amino-reactive p-nitrophenyl ester moiety includep-nitrophenyl iodoacetate (NPIA).

Compositions

The present invention provides compositions, including pharmaceuticalcompositions, comprising a subject polypeptide-polymer conjugate.

In some embodiments, a subject composition comprises a subjectpolypeptide-polymer conjugate, wherein the subject polypeptide-polymerconjugate is homogeneous, e.g., all of the polypeptides of thepolypeptide-polymer conjugate comprise the same amino acid sequence. Forexample, in some embodiments, a subject composition comprises aplurality of (e.g., multiple copies of) a subject polypeptide-polymerconjugate, where each polypeptide-polymer conjugate molecule comprisespolypeptides that all have the same amino acid sequence.

In other embodiments, a subject composition comprises two or morespecies of a subject polypeptide-polymer conjugate, e.g., a subjectcomposition comprises a first polypeptide-polymer conjugate, where thefirst polypeptide-polymer conjugate comprises polypeptides of a firstamino acid sequence; and at least a second polypeptide-polymerconjugate, wherein the second polypeptide-polymer conjugate comprisespolypeptides of a second amino acid sequence that is different from thefirst amino acid sequence. In some embodiments, a subject compositioncomprises a third or additional polypeptide-polymer conjugates. As onenon-limiting example, a first polypeptide-polymer conjugate comprises afirst polypeptide that provides for binding to an integrin; and a secondpolypeptide-polymer conjugate that comprises a second polypeptide thatactivates a cell signaling pathway. Various other combinations of first,second, etc., polypeptides can be used. The ratio of the firstpolypeptide-polymer conjugate to the second polypeptide-polymerconjugate in a subject composition can be varied, e.g., from about 0:001to 10³ to about 10³ to 0.001. Similarly, where a subject compositioncomprises a first, a second, and a third polypeptide-polymer conjugate,the ratios of the first, second, and third polypeptide-polymerconjugates can be varied.

A subject composition can comprise, in addition to a subjectpolypeptide-polymer conjugate, one or more of: a salt, e.g., NaCl, MgCl,KCl, MgSO₄, etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; and the like.

The present invention provides compositions comprising a subjectpolypeptide-polymer conjugate and a pharmaceutically acceptableexcipient. Suitable excipient vehicles are, for example, water, saline,dextrose, glycerol, ethanol, or the like, and combinations thereof. Inaddition, if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of the agent adequate to achieve thedesired state in the subject being treated. The pharmaceuticallyacceptable excipients, such as vehicles, adjuvants, carriers ordiluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

As used herein, the terms “pharmaceutically acceptable carrier” and“pharmaceutically acceptable excipient” are used interchangeably, andinclude any material, which when combined with a subjectpolypeptide-polymer conjugate does not substantially affect thebiological activity of the conjugate, does not induce an immune responsein a host, and does not have any substantial adverse physiologicaleffect on the host. Examples include, but are not limited to, any of thestandard pharmaceutical carriers such as a phosphate buffered salinesolution, water, emulsions such as oil/water emulsion, and various typesof wetting agents. Other carriers may also include sterile solutions,tablets including coated tablets and capsules. Typically such carrierscontain excipients such as starch, milk, sugar, certain types of clay,gelatin, stearic acid or salts thereof, magnesium or calcium stearate,talc, vegetable fats or oils, gums, glycols, or other known excipients.Such carriers may also include flavor and color additives or otheringredients. Compositions comprising such carriers are formulated bywell known conventional methods.

The pharmaceutical compositions may be formulated for a selected mannerof administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, intratumoral, peritumoral,subcutaneous, or intramuscular administration. For parenteraladministration, such as subcutaneous injection, the carrier can comprisewater, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactate polyglycolate)may also be employed as carriers for a subject pharmaceuticalcomposition. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

In some embodiments, a subject pharmaceutical composition isadministered parenterally, e.g., intravenously. Thus, the inventionprovides compositions for parenteral administration which comprise asubject conjugate dissolved or suspended in an acceptable carrier,preferably an aqueous carrier, e.g., water, buffered water, saline,phosphate-buffered saline, and the like. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

A subject composition can be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionscan be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations can range from 3 and 11,e.g., from about pH 5 to about pH 9, or from about pH 7 to about pH 8.

Implantable Tissues and Devices

In some embodiments, a subject polypeptide-polymer conjugate is coatedonto, layered onto, incorporated into, or forms, an implantable tissueor device, e.g., an artificial tissue; an implant into a tissue; acoating for an implantable device (such as an intravascular stent, anartificial joint, a scaffold, a graft (e.g., an aortic graft), anartificial heart valve, a cerebrospinal fluid shunt, a coronary shunt, apacemaker electrode, an endocardial lead, etc.); an implantable drugdelivery system; and the like. Artificial tissues include, e.g.,synthetic heart valves (e.g., a synthetic aortic valve, a syntheticmitral valve, etc.). Stents include, e.g., self-expandable stents,balloon-expandable stents, and stent-grafts. Biomaterials include, e.g.,films, gels, sponges, gauzes, nonwoven fabrics, membranes, microspheres,microcapsules, threads, guide channels, and the like.

For example, in some embodiments, a subject polypeptide-polymerconjugate is layered or coated onto or otherwise attached to a matrix,to form a synthetic implantable device. For example, a matrix (alsoreferred to as a “biocompatible substrate”) is a material that issuitable for implantation into a subject and onto which a subjectpolypeptide-polymer conjugate is layered, coated, or otherwise attached.A biocompatible substrate does not cause toxic or injurious effects onceimplanted in the subject. In one embodiment, the biocompatible substrateis a polymer with a surface that can be shaped into the desiredstructure that requires repairing or replacing. The biocompatiblesubstrate can also be shaped into a part of a structure that requiresrepairing or replacing. The biocompatible substrate provides thesupportive framework onto which a subject polypeptide-polymer conjugatecan be layered, coated, or otherwise attached.

In some embodiments, a matrix or a scaffold comprising attached theretoa subject polypeptide-polymer conjugate further comprises one or morecells and/or one or more cell types bound to the matrix or scaffoldcomprising the polypeptide-polymer conjugate. Such matrices or scaffoldsare useful in the context of tissue engineering, cell culturing, celltransplantation, etc.

In some embodiments, a drug delivery device comprises a subjectpolypeptide-polymer conjugate. For example, the drug release device canbe based upon a diffusive system, a convective system, or an erodiblesystem (e.g., an erosion-based system). For example, the drug releasedevice can be an electrochemical pump, osmotic pump, an electroosmoticpump, a vapor pressure pump, or osmotic bursting matrix, e.g., where thedrug is incorporated into a polymer and the polymer provides for releaseof drug formulation concomitant with degradation of a drug-impregnatedpolymeric material (e.g., a biodegradable, drug-impregnated polymericmaterial). In other embodiments, the drug release device is based uponan electrodiffusion system, an electrolytic pump, an effervescent pump,a piezoelectric pump, a hydrolytic system, etc.

In some embodiments, the implantable drug delivery system isprogrammable to provide for administration of an active agent. Exemplaryprogrammable, implantable systems include implantable infusion pumps.Exemplary implantable infusion pumps, or devices useful in connectionwith such pumps, are described in, for example, U.S. Pat. Nos.4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276;6,241,704; 6,464,687; 6,475,180; and 6,512,954. A further exemplarydevice is the Synchromed infusion pump (Medtronic).

An implantable drug delivery device can be used to delivery any of avariety of agents, e.g., immune response modifiers, anti-proliferatives,anti-apoptotic agents, anti-mitotic agents, anti-platelet agents,platinum coordination complexes, hormones, anticoagulants, fibrinolyticagents, anti-secretory agents, anti-migratory agents,immunosuppressives, angiogenic agents, angiotensin receptor blockers,nitric oxide donors, antisense oligonucleotides, cell cycle inhibitors,corticosteroids, angiostatic steroids, anti-parasitic drugs,anti-glaucoma drugs, antibiotics, differentiation modulators, antiviraldrugs, anticancer drugs, and anti-inflammatory drugs.

Utility

A subject polypeptide-polymer conjugate finds use in variousapplications, including therapeutic (e.g., drug delivery, implantabledevices, tissue engineering, regenerative medicine), diagnostic, drugdiscovery, and research applications.

Therapeutic Applications

A subject polypeptide-polymer conjugate finds use in various therapeuticapplications.

For example, as discussed above, a subject polypeptide-polymer conjugatecan be attached to a drug delivery device, where the biologically activepolypeptide component of the polypeptide-polymer conjugate confers afunctionality, and where the drug delivery device provides a therapeuticagent. For example, the biologically active polypeptide could providetargeting to a particular cell type or tissue type in need of treatmentwith a therapeutic agent, and the drug delivery device could provide thetherapeutic agent in a localized manner.

As another example, the biologically active polypeptide component of asubject polypeptide-polymer conjugate could itself be a therapeuticagent, e.g., by providing for induction of apoptosis in a tumor cell; byinducing coagulation of blood at a treatment site; by inhibitingplatelet aggregation; by inducing angiogenesis; by inducing celldifferentiation; and the like.

As another example, as discussed above, a subject polypeptide-polymerconjugate can be attached to an implantable medical device, e.g., astent, a shunt, an artificial valve, a lead, an artificial joint, agraft, an electrode, etc., where the biologically active polypeptidecomponent of the subject polypeptide-polymer conjugate provides adesired activity, e.g., reduction of neointimal hyperplasia restenosis;inhibition of cell proliferation; inhibition of cell adhesion; and thelike.

As another example, as discussed above, a subject polypeptide-polymerconjugate can be attached a matrix or a scaffold, where thepolypeptide-polymer conjugate provides for cell binding. The matrix orscaffold comprising a subject polypeptide-polymer conjugate with orwithout cells bound to the polypeptide-polymer conjugate can beintroduced into an individual in the context of cell transplantation,tissue engineering, etc.

In some embodiments, a subject polypeptide-polymer conjugate finds usein inducing angiogenesis (e.g., where the polypeptide is one thatinduces angiogenesis) in an individual in need thereof, e.g., in or nearan ischemic tissue.

Research Applications

A subject polypeptide-polymer conjugate finds use in various researchapplications, e.g., to investigate a cell signaling pathway; and thelike. A subject polypeptide-polymer conjugate can be administered to anexperimental non-human animal model of a disease, to test the effect ofthe subject polypeptide-polymer conjugate on a physiological response inthe model. A subject polypeptide-polymer conjugate can also be used indrug screening applications.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Synthesis and Characterization of Polypeptide-PolymerConjugates

A potently active multivalent form of the protein Sonic hedgehog (Shh)was produced by bioconjugation of a modified recombinant form of Shh tothe linear polymers polyacrylic acid (pAAc) and hyaluronic acid (HyA)via a two step reaction exploiting carbodiimide and maleimide chemistry.Efficiency of the conjugation was ˜75% even at stoichiometric ratios of30 Shh molecules per linear HyA chain (i.e., 30:1 Shh:HyA). Bioactivityof the conjugates was tested via a cellular assay across a range ofstoichiometric ratios of Shh molecules to HyA linear chains, which wasvaried from 0.6:1 Shh:HyA to 22:1 Shh:HyA. Results indicate that lowconjugation ratios decrease Shh bioactivity and high ratios increasethis activity beyond the potency of monomeric Shh, with approximatelyequal activity between monomeric soluble Shh and conjugated Shh at 7:1Shh:HyA. In addition, high ratio constructs increased angiogenesisdetermined by the in vivo chick chorioallantoic membrane (CAM) assay.These results are captured by a kinetic model of multiple interactionsbetween the Shh:HyA conjugates and cell surface receptors resulting inhigher cell signaling at lower bulk Shh concentrations.

Methods Recombinant Shh and Bioconjugation Techniques

Using the cDNA of the N-terminal signaling domain of rat Shh previouslydescribed (15), base pairs coding for an additional cysteine residue anda 6× His tag were added through PCR onto the C-terminus of the proteinto allow for sulfhydryl-based reactions and protein purification,respectively. This tethering site was specifically chosen based onstudies demonstrating that this area of the protein is distant from itsactive site, and inert molecules attached here do not alter activity(16). The produced modified Shh PCR product was inserted into apBAD-HisA (Invitrogen, Carlsbad, Calif.) plasmid, the resulting plasmidwas confirmed by DNA sequencing, and the protein expressed in BL21(DE3).pLys.E E. coli through arabinose induction. After induced proteinexpression, cells were lysed, and the resulting expressed Shh purifiedusing NiNTA (Qiagen, Valencia, Calif.) binding followed by imidazoleelution. The purified protein was dialyzed into pH 7.4 PBS containing10% glycerol, 2 mM EDTA, and 50 μM ZnSO₄.

Purified Shh was conjugated to linear polymers through a 2-step reactionusing carbodiimide chemistry at the carboxylate group of the polymer anda maleimide reaction at the protein C-terminal cysteine (FIG. 1). Thefirst step was the addition of [N-ε-maleimidocaproic acid] hydrazide(EMCH, Pierce Biotechnology, Rockford, Ill.) to the linear polymer toallow for the subsequent attachment of the protein. This non-immunogenichydrazide—maleimide hetereobifunctional crosslinker was added to the twolinear polymers using the same general procedure, but with slightlydifferent reaction conditions. For pAAc conjugates, 450,000 Da pAAc(Polysciences, Warrington, Pa.) at 2 mg/ml was reacted with1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) at 3.9mg/ml, N-hydroxysulfosuccinimide (sulfo-NHS) at 1.1 mg/ml, and 0.5 mg/mlEMCH at room temperature for 2 hours in pH 6.5 MES buffer as describedfor the attachment of small peptide sequences (17). For the activationof HyA, a method similar to that previously described for the attachmentof hydrazides (9) was used with 10⁶ Da MW HyA. (Genzyme, CambridgeMass.) This was dissolved and reacted at 3 mg/ml with the sameconcentrations of EDC, sulfo-NHS, and EMCH used in the pAAc reactionovernight in 0.1 M MES buffer, pH 5.0. After the attachment of the EMCH,the resulting maleimide activated linear polymers were separated fromthe unconjugated reagents through sequential dilution and centrifugationin 50,000 MW cutoff centrifuge filters (Pall Gellman).

The activated polymers were then reacted with the Shh in varyingstoichiometric feed ratios to produce conjugates of varying molecularsubstitution. This reaction was performed at 4° C. overnight in 0.1 MMES buffer (pH 6.5) containing 50 μM Tris (2-carboxyethyl) phosphinehydrochloride (TCEP, Pierce Biotechnology, Rockford, Ill.) to keep theC-terminus Shh cysteine reduced for duration of the reaction. After thereaction, any remaining maleimide groups on the linear polymer werereduced by the addition of 0.5 mM dithiothreitol and incubation at 4° C.for 1 hr.

All conjugation reactions were assayed by gel electrophoresis, comparingreaction solutions to an equal mass of unreacted Shh to visually inspectprotein coupling efficiency. In addition, sets of triplicate Shh:HyAconjugation reactions at 20:1 and 10:1 molar feed ratios of Shh to HyAwere dialyzed overnight in 0.1 MES buffer (pH 6.5) using Spectra/Por®Float-A-Lyzer® devices (Spectrum Laboratories, Rancho Dominguez, Calif.)to remove non-conjugated Shh. Protein concentrations in the dialyzedHyA-Shh solutions were then quantified using a microBCA assay (PierceBiotechnology, Rockford, Ill.).

Bioactivity Assay

In order to test bioactivity, murine embryonic C3H10T1/2 cells (AmericanType Culture Collection, Manassas Va.) were induced to differentiateinto an osteogenic line by exposure to Shh as described elsewhere (18,19). Briefly, the cells were plated at 5000 cells/well in 96 well platesin normal growth media (αMEM with 10% FBS). After 2 days, the medium wasreplaced with a low FBS (2%) media and supplemented with the proteinsand conjugate reaction solutions. Test conditions included soluble Shhin the range of 1-100 nM, soluble Shh in the same range along withunconjugated HyA at 50 μg/ml, or the Shh:HyA conjugate in quantitiessuch that the concentration of Shh in the media solutions were also1-100 nM. After incubation for an additional 3 days, the cells werewashed and lysed, and the cell lysate was assayed for differentiation bymeasuring alkaline phosphatase (ALP) activity using the fluorescentprobe 9-H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO,Molecular Probes, Eugene Oreg.). Unconjugated polyacrylic acid was shownto inhibit the differentiation of the cells, so bioactivity testing ofthese conjugates was not performed.

Angiogenesis Assays

Shh is a known angiogenic agent (20). Induction of angiogenesis fromsoluble Shh and Shh:HyA conjugates was assayed using a CAM window assay.Fertile white leghorn eggs (Charles River, Franklin Conn.) wereincubated at 37° C. in a humidified environment until day 8, at whichtime 2 ml of albumin was removed from the blunt end of the egg, and asmall 1 cm×1 cm window was made in the shell on the opposite side.Sterile squares of filter paper loaded with sterile PBS, 0.1 μg of Shh,or 0.1 μg Shh of the 20:1 Shh:HyA feed ratio conjugate were placeddirectly on the developing CAM. This window was then sealed withparafilm and the eggs returned to the incubator. Angiogenesis around thetest materials was microscopically evaluated 3 days later using anOlympus SZX7 stereoscope. High resolution photomicrographs were takenusing an attached Qlmaging Qfire camera. These images were analyzedusing ImageJ software to quantify the number of blood vessels per unitlength in a square perimeter surrounding the implants at distances of0.1 and 0.25 cm away from its edge. Linear density measurements for eachgroup were tested for statistical significance using a one-way ANOVA onboth the 0.1 and 0.25 cm distance data, followed by pairwise Holm'st-tests of the individual groups.

Molecular Modeling of Shh:HyA Conjugate Cell Signaling

Binding and trafficking numerical models that describe expression of Glitranscriptional effectors in response to monomeric Shh (21, 22) andnumerical kinetic models describing multivalent ligand-receptor binding(23) (FIG. 3) were built upon to model Shh:HyA conjugate cell signaling.In FIG. 3, the Shh core signaling network and hypothesized reactionsinvolving a multivalent conjugate are shown around a representativecell. Arrows between proteins represent binding or dissociation, arrowsfrom genes to proteins represent expression, and arrows from proteins togenes indicate activation or repression. Smo, Smoothened. At thecellular level, Shh induces cell fate switching by interacting with itstransmembrane receptor, Patched (Ptc). In absence of Shh, Ptc repressesthe signaling activity of the transmembrane protein Smo and thereforeacts as a repressor of Shh signaling as described previously (Lai etal., 2004). gli upregulation represents positive feedback, whereas ptcupregulation yields negative feedback. Simulations explore the effect ofvarious mechanisms: binding of HyA:Shh conjugate (avidity);internalization of conjugate-Ptc complexes; and degradation of HyA:Shh.

Nondimensional equations for Shh signaling are shown below:

For Soluble Shh Signal Transduction

$\frac{\partial B}{\partial\tau} = {{\alpha_{off}B} + {\alpha_{on}{AD}} - {\beta_{i\; n}B} + {\gamma_{out}C}}$$\frac{\partial C}{\partial\tau} = {{\beta_{i\; n}B} - {\gamma_{out}C} - {\Theta_{C}C}}$$\frac{\partial D}{\partial\tau} = {{\alpha_{off}B} - {\alpha_{on}{AD}} + {\alpha_{p}{Promoter}} + {\beta_{p}{Basal}} + ɛ_{out} - {\delta_{i\; n}D}}$$\frac{\partial E}{\partial\tau} = {{{- ɛ_{out}}E} + {\delta_{i\; n}D} - {\Theta_{E}E}}$AP = k_(AP)G₁$\frac{\partial G_{1}}{\partial\tau} = {{\alpha \; {Promoter}} + {\beta \; {Basal}} - G_{1}}$$\frac{\partial G_{3}}{\partial\tau} = {\frac{\gamma}{G_{1}} - {G_{3}*\left( \frac{\delta}{{{Kg}\; 3{rc}} + {Signal}} \right)} - G_{3}}$$\frac{\partial G_{3\; R}}{\partial\tau} = {{G_{3}*\left( \frac{\delta}{{{Kg}\; 3{rc}} + {Signal}} \right)} - G_{3\; R}}$${Signal} = \frac{1}{1 + {ϛ\; D}}$

For Conjugate Signal Transduction Replace

$\frac{\partial B}{\partial\tau}$

expression above with the following expressions for cell surfacemultivalent-receptor complexes of valency i (Bcom_(i)) and maximumvalency of f:

  for  i = 1$\frac{\partial{Bcom}_{1}}{\partial\tau} = {{2\alpha_{off}{Bcom}_{2}} - {k_{x}{\alpha_{on}\left( {f - 1} \right)}{Bcom}_{1}D} - {\alpha_{on}{AD}} - {\alpha_{{off}\;}{Bcom}_{1}} + {\alpha_{on}{AD}} - {\beta_{i\; n}{Bcom}_{1}} + {\gamma_{out}{Ccom}_{1}}}$  for  i = [2, f − 1]$\frac{\partial{Bcom}_{i}}{\partial\tau} = {{{- i}\; \alpha_{off}{Bcom}_{i}} + {k_{x}{\alpha_{on}\left( {f - i + 1} \right)}{Bcom}_{i - 1}D} - {\left( {f - i} \right)k_{x}\alpha_{on}{Bcom}_{i}D} + {\left( {i + 1} \right)\alpha_{off}{Bcom}_{i + 1}} - {\beta_{i\; n}{Bcom}_{i}} + {\gamma_{out}{Ccom}_{i}}}$  for  i = f$\frac{\partial{Bcom}_{f}}{\partial\tau} = {{{- f}\; \alpha_{off}{Bcom}_{f}} + {k_{x}\alpha_{on}{Bcom}_{f - 1}D} - {B_{i\; n}{Bcom}_{f}} + {\gamma_{out}{Ccom}_{f}}}$

Replace

$\frac{\partial C}{\partial\tau}$

expression above with the following expressions for internalizedmultivalent-receptor complexes of valency i (Ccom_(i)):

for  i = [1, f]$\frac{\partial{Ccom}_{i}}{\partial\tau} = {{\beta_{i\; n}{Bcom}_{i}} - {\gamma_{out}{Ccom}_{i}} - {\Theta_{C}{Ccom}_{i}}}$

Replace

$\frac{\partial D}{\partial\tau}$

expression above with the following:

$\frac{\partial D}{\partial\tau} = {{\alpha_{off}{\sum\limits_{1}^{f}{(i){Bcom}_{i}}}} - {k_{x}\alpha_{on}{\sum\limits_{1}^{f - 1}{\left( {f - i} \right){Bcom}_{i}}}} - {\alpha_{on}{AD}} + {\alpha_{p}{Promoter}} + {\beta_{p}{Basal}} + {ɛ_{out}E} - {\delta_{i\; n}D}}$

Initial conditions, parameters, and variable descriptions are listedwith their literature sources in Table 1. “Promoter” and “Basal” termshave been previously defined (Lai, Robertson et al. 2004). See Saha andSchaffer Development, 2006 for sensitivity analysis of parameters in thesoluble Shh network.

To develop the simplest model for the C3H10T1/2 bioactivity data in FIG.4, the following assumptions were invoked: Patched (Ptc) repression ofSmoothened is not affected by the Ptc receptor aggregation;ligand-induced internalization rate is the same for all valencies;alkaline phosphatase activity is linearly proportional to Gli1 levels ina cell; and differentiation of a C3H10T1/2 cell does not change itsresponsiveness to Shh. Initial binding of the HyA:Shh conjugate wasassumed to follow monomeric Shh-Ptc binding rates, but all otheradditional binding of Shh moieties from the conjugate to other Ptcreceptors were assumed to occur at a higher rate. This assumption hasbeen called the equivalent site hypothesis to take into account theacceleration of binding after initial binding of a multivalent conjugate(24).

Model parameters were taken from literature (21, 22); however, a numberof parameters were estimated directly from the bioactivity data in FIG.4. First, the monomeric Shh-Ptc binding constant k_(on)/k_(off) and thealkaline phosphatase activity:Gli1 expression ratio were estimated fromthe soluble Shh bioactivity curve. In addition, the multimeric Shh-Ptcbinding constant was directly estimated from the 22:1 conjugate curve(See Table 1). Parameters were taken either from Shh literature or fromsimilar ligand-receptor systems. All cellular rate constants areaveraged within the volume or surface of the cell, since these constantsare not known to vary spatially within a cell. The initial conditionsfor the simulation results shown in FIG. 7 are listed with eachvariable. A convenient way to understand the relative importance ofevery term in the differential equations is to compare nondimensionalconcentrations and parameters in Table 1.

TABLE 1 Parameters definitions and value ranges. Non- Dimensional Value/dimensional Value/ Parameter Description Range Source Parameter RangeNondimensionalization Core Signaling Pathway [Shh] Extracellular Initialvariable A Initial (mol membranic Shh Condition = 0 Condition = 0Shh)/(L of concentration extracellular liquid volume)/(Kgli3)/ (vff)[PtcShh_(in)] Extracellular Initial variable B Initial (mol membranicPtc- PtcShh Condition = 0 Condition = 0 Shh complex)/(L of concentrationextracellular liquid volume)/(Kgli3)/ (vff) [PtcShh_(out)] IntracellularInitial variable C Initial (mol intracellular PtcShh Condition = 0Condition = 0 Ptc-Shh complex)/ concentration (L of intracellular liquidvolume)/ (Kgli3) [Ptc_(out)] Extracellular Initial variable D Initial(mol membranic free Ptc Condition = Condition = Ptc)/(L of concentration2.0 nM 0.605 extracellular liquid volume)/(Kgli3)/ (vff) [Ptc_(in)]Intracellular Initial variable E Initial (mol intracellular PtcCondition = Condition = Ptc-Shh complex)/ concentration 0.33 nM 0.402 (Lof intracellular liquid volume)/ (Kgli3) [Gli1] Intracellular Initialvariable G₁ Initial (mol intracellular Gli1 Condition = Condition =Gli1)/(L of concentration 1.63 nM 1.97 intracellular liquidvolume)/(Kgli3) [Gli3] Intracellular Initial variable G₃ Initial (molintracellular Gli3 Condition = Condition = Gli3)/(L of concentration5.81 nM 7.00 intracellular liquid volume)/(Kgli3) [Gli3R] IntracellularInitial variable G_(3R) Initial (mol intracellular Gli3 Condition =Condition = Gli3 Repressor)/(L Repressor 61.2 nM 18.44 of intracellularliquid concentration volume)/(Kgli3) vf Void fraction 0.2 (Lauffenburgerof tissue and Linderman 1993) vff intracellular 4 (1 − vf)/vf volume/extracellular volume k_(deg) Degradation 0.009 min⁻¹ (Chen, rateconstant Kessler et for Gli1 al. 1999) K_(Gli3) Dissociation 8.3 ×10⁻¹⁰M (Lai, constant for Robertson Gli3 binding et al. 2004) to Gli1DNA binding site K_(shh) Dissociation 8.5 × 10⁻¹⁰M (Fuse, constant forfor model Maiti et al. Shh-Ptc without 1999; binding sterics; 4.5 ×Taipale, 10⁻⁹M for Cooper et model with al. 2002) sterics k_(off)Dissociation 0.10 min⁻¹ 0.3 min⁻¹ α_(off) 11 k_(off)/k_(deg) of Shh fromfor EGF Ptc (Lauffenburger and Linderman 1993) k_(on) Association of120,000,000M⁻¹ k_(off)/K_(shh) α_(on) 44.44 k_(on) * (K_(Gli3) *vff)/k_(deg) Shh with Ptc min⁻¹ for model without sterics; 22,666,667M⁻¹min⁻¹ for model with sterics k_(Cdeg) Degradation 0.00198 min⁻¹ 0.0022min⁻¹ Θ_(C) 0.220 k_(Cdeg)/k_(deg) rate constant for for EGFintracellular (Lauffenburger Shh-Ptc and complex Linderman 1993) k_(Pin)Import to 0.03 min⁻¹ for EGFR δ_(in) 3.33 k_(Pin)/k_(deg) endosome of(Lauffenburger surface free and receptors Linderman 1993) k_(Pout)Recycle to 0.00036 min⁻¹ 0.058 min⁻¹ ϵ_(out) 0.0403 k_(Pout)/k_(deg)surface of (Lauffenburger endosomal and free receptors Linderman 1993);0.003 min⁻¹ for Dpp (Lander, Nie et al. 2002) k_(Cin) Import to 0.2min⁻¹ 0.03-0.3 min⁻¹ β_(in) 33.3 k_(Cin)/k_(deg) endosome of for surfaceShh- EGF bound (Lauffenburger complexes and Linderman 1993) k_(Cout)Export to 0.00181 min⁻¹ 0.00402 min⁻¹ γ_(out) 0.20 k_(Cout)/k_(deg)surface of for intracellular Dpp Shh-bound (Lander, complexes Nie et al.2002) k_(Gmax) Maximum 1.99 × 10⁻¹⁰M 2.4 × 10⁻¹⁰M α 30.4k_(Gmax)/(K_(Gli3) * k_(deg)) rate of Gli min⁻¹ min⁻¹ synthesis (Lai,Robertson et al. 2004) k_(Gbas) Basal rate of 1.53 × 10⁻¹²M k_(Gmax)/130β 0.233 k_(Gbas)/(K_(Gli3) * k_(deg)) Gli synthesis min⁻¹ r_(g3b) Basalrate of 3.1 × 10⁻¹⁹M² 1.6 × 10⁻¹⁹M² γ 50.0 r_(g3b)/(K_(Gli3) *K_(Gli3) * Gli3 synthesis min⁻¹ min⁻¹ k_(deg)) (Lai, Robertson et al.2004) k_(Pdeg) Degradation 0.09 min⁻¹ 0.045-0.071 min⁻¹ Θ_(E) 10.0k_(Pdeg)/k_(deg) rate constant (French for Ptc and Lauffenburger 1996);0.006 min⁻¹ (Lander, Nie et al. 2002) K_(ptc) Half-maximal 3.32 × 10⁻¹¹M8.3 × 10⁻¹¹M ζ 2.50 K_(Gli3)/K_(ptc) conc for Ptc (Taipale, whichinhibits Cooper et Smo signaling al. 2002) k_(g3r) Rate constant 0.0117min⁻¹ 0.0117 min⁻¹ δ 1.30 k_(g3r)/k_(deg) for the (Lai, conversion ofRobertson Gli3 to Gli3R et al. 2004) k_(Pmax) Maximum 1.50 × 10⁻¹⁰M 7.5× 10⁻¹⁰M α_(P) 5.01 k_(Pmax)/ rate of Ptc min⁻¹ min⁻¹ (K_(Gli3) * vff *k_(deg)) synthesis (Lai, Robertson et al. 2004); Set from soluble curvein FIG. 3 k_(Pbas) Basal rate of 1.15 × 10⁻¹²M k_(Pmax)/130 β_(P) 0.0385k_(Pbas)/(K_(Gli3) * vff * k_(deg)) Ptc synthesis min⁻¹ K_(g3rc)Sensitivity 0.12 0.1 (Lai, constant of Robertson the conversion et al.2004) to signal strength bc Binding 1 (Keller cooperativity 1995) tcTranscriptional 0.5 (Keller efficiency 1995) r Transcriptional 0.2 (Lai,repression Robertson et al. 2004) Afr Affinity ratio 0.5 between Gli1and Gli3 for DNA binding site k_(Ap) Ratio of Gli1 9.3 Set from proteinto soluble RFU units of curve in Alkaline FIG. 3 Phosphatase activityMultivalent Conjugate Reactions [PtcShh_(i,in)] Extracellular Initialvariable Bcom_(i) Initial (mol membranic Ptc- PtcShh Condition = 0Condition = 0 Shh_(i) complex)/(L of concentration extracellular liquidof valency i volume)/(Kgli3)/ (vff) [PtcShh_(i,out)] IntracellularInitial variable Ccom_(i) Initial (mol intracellular PtcShh Condition =0 Condition = 0 Ptc-Shh_(i) complex)/ concentration (L of intracellularof valency i liquid volume)/ (Kgli3) i Valency index 1, 2, 3, . . . f tomaximum valency of f k_(x) Factor by 12 Set from which binding 22:1curve of second and in FIG. 3 other Shh moeties on conjugate bind to Ptc

An alternative model that incorporates steric hindrance of HyA chains asa simple reduction in conjugate binding affinity to Ptc was alsoformulated. For the alternative model incorporating sterics, termed the“model with sterics,” the multimeric Shh-Ptc binding constant k_(on) for0.6:1, 3.5:1, and 7:1 conjugation feed ratios was reduced 5.5 fold tomatch the experimental data from the 0.6:1 curve in FIG. 4. Below is theBERKELEY MADONNA code for the simulations for a multivalent conjugate,where f=5 (5:1 Shh:HyA conjugate). The code below is termed “modelwithout sterics” in the main text. As mentioned in the Methods section,for the “model with sterics,” only one parameter change was made: themultimeric Shh-Ptc binding constant k_(on) was reduced 5.5 fold to matchthe experimental data from the 0.6:1 curve in FIG. 4.

METHOD RK4 STARTTIME = 0 STOPTIME=500 DT = 0.0000002 Shh=S*Kshh ;---------------------------------------------------------------------------------------------------;Define Promoter and Basal variable from Lai et. al. Biophys J. 2004 ;K1 = equilibrium dissociation binding constant of Gli1 ; K2 =equilibrium dissociation binding constant of Gli3 ; afr = affinity ratio= K2/K1 ; bc = binding cooperativity ; tc = transcriptionalcooperativity ; r = repression ratio ;---------------------------------------------------------------------------------------------------; Gli Core Promoter and Basal expressions from Lai et. al. Biophys J.2004 Promoter=((afr*G1 + G3)*(afr{circumflex over ( )}2*bc{circumflexover ( )}2*G1{circumflex over ( )}2 + 3*tc{circumflex over ( )}2 +3*bc*tc*(G3 + 2*G3R*r*tc) + afr*bc*G1*(2*bc*G3 + 3*tc + 3*bc*G3R*r*tc) +bc{circumflex over ( )}2*(G3{circumflex over ( )}2 + 3*G3*G3R*r*tc +3*G3R{circumflex over ( )}2*r{circumflex over ( )}2*tc{circumflex over( )}2)))/(1 + afr{circumflex over ( )}3*bc{circumflex over( )}2*G1{circumflex over ( )}3 + bc{circumflex over ( )}2*G3{circumflexover ( )}3 + 3*G3R + 3*bc*G3R{circumflex over ( )}2 + bc{circumflex over( )}2*G3R{circumflex over ( )}3 + 3*bc*G3{circumflex over ( )}2*(1 +bc*G3R) + 3*G3*(1 + bc*G3R){circumflex over ( )}2 + 3*afr{circumflexover ( )}2*bc*G1{circumflex over ( )}2*(1 + bc*(G3 + G3R)) +3*afr*G1*(1 + bc*(G3 + G3R)){circumflex over ( )}2) Basal=((1 +afr{circumflex over ( )}3*bc{circumflex over ( )}2*G1{circumflex over( )}3 + bc{circumflex over ( )}2*G3{circumflex over ( )}3 + 3*G3R*r +3*bc*G3R{circumflex over ( )}2*r{circumflex over ( )}2 + bc{circumflexover ( )}2*G3R{circumflex over ( )}3*r{circumflex over ( )}3 +3*bc*G3{circumflex over ( )}2*(1 + bc*G3R*r) + 3*G3*(1 +bc*G3R*r){circumflex over ( )}2 + 3*afr{circumflex over( )}2*bc*G1{circumflex over ( )}2*(1 + bc*(G3 + G3R*r)) + 3*afr*G1*(1 +bc*(G3 + G3R*r)){circumflex over ( )}2))/(1 + afr{circumflex over( )}3*bc{circumflex over ( )}2*G1{circumflex over ( )}3 + bc{circumflexover ( )}2*G3{circumflex over ( )}3 + 3*G3R + 3*bc*G3R{circumflex over( )}2 + bc{circumflex over ( )}2*G3R{circumflex over ( )}3 +3*bc*G3{circumflex over ( )}2*(1 + bc*G3R) + 3*G3*(1 +bc*G3R){circumflex over ( )}2 + 3*afr{circumflex over( )}2*bc*G1{circumflex over ( )}2*(1 + bc*(G3 + G3R)) + 3*afr*G1*(1 +bc*(G3 + G3R)){circumflex over ( )}2) ;---------------------------------------------------------------------------------------------------;Define dimensional constants for Shh binding and transport equations ;---------------------------------------------------------------------------------------------------koff=0.1 ; dissociation of Shh from Ptc (min−1) kdegc=0.00198 ;Degradation rate constant for Shh-Ptc complex (min−1) kp=0.03 ;Lauffenburger for EGF keR=3e−2 min−1 (p95) kq=0.00036 ; Lauffenburgerfor EGF krec=5.8e−2 min−1 (p95) kin=0.021 ; Lauffenburger for EGFkeC=0.03-0.3 min−1 (p95) kout=0.00181 ; Export to surface ofintracellular Shh-Ptc complex (min−1) kg=0.09 ; Degradation rateconstant for Ptc (.045-0.071 min−1) kon=koff/Kshh ; Association of Shhwith Ptc vf=0.2 ; void fraction of tissue vff=(1−vf)/vf ; void fractionfactor = intracellular volume/extracellular volume Do=2.0e−9 ; initialfree Ptc concentration for slider in (mol membranic free Ptc) / (L ofextracellular liquid volume) Eo=0.33e−9 ; initial internal Ptcconcentration for slider in (mol internal Ptc) / (L of intracellularliquid volume) ;---------------------------------------------------------------------------------------------------;Define nondimensional constants ; time in units of 1/kdeg, Degradationrate constant for Gli 1 (.009 min−1) ;---------------------------------------------------------------------------------------------------theta_e=kg/kdeg alph_off=koff/kdeg alph_on=kon*(Kgli3*vff)/kdegbeta_in=kin/kdeg gmma_out=kout/kdeg theta_c=kdegc/kdegalph_p=kcatp/(Kgli3*vff*kdeg) beta_p=rbas/(Kgli3*vff*kdeg)eps_out=kq/kdeg dlta_in=kp/kdeg ce=Kgli3/Kptc ;*******************************************************************************;Define Nondimensional Shh binding equations ; Nondimensional variables:;A = (mol Shh) / (L of extracellular liquid volume) / (Kgli3) / (vff) ;B= (mol membranic Ptc-Shh complex) / (L of extracellular liquid volume) /(Kgli3) / (vff) ;C = (mol intracellular Ptc-Shh complex) / (L ofintracellular liquid volume) / (Kgli3) ;D = (mol membranic free Ptc) /(L of extracellular liquid volume) / (Kgli3) / (vff) ;E = (molintracellular Ptc-Shh complex) / (L of intracellular liquid volume) /(Kgli3) ; Multimeric model from Lauffenberger 1993; Perelson 1986;Bcom[i] = (mol membranic Ptc-Shh[i] complex) / (L of extracellularliquid volume) / (Kgli3) / (vff) ;Ccom[i] = (mol intracellularPtc-Shh[i] complex) / (L of intracellular liquid volume) / (Kgli3) ;*******************************************************************************d/dt (A) = 0 d/dt (D) = (alph_p)*Promoter + (beta_p)*Basal + (eps_out)*E− (alph_on)*A*D+k_x*Sum2− kx*Sum1*D − (dlta_in)*D d/dt (E) =(dlta_in)*D−(eps_out)*E−(theta_e)*E sum1v[1..(f−1)]=(f−i)*Bcom[i]sum1=arraysum(sum1v[*]) sum2v[1..f]=(i)*Bcom[i] sum2=arraysum(sum2v[*])f=5 ; maximum valency INIT Bcom[1..f] = 0 d/dt (Bcom[1]) = (alph_on)*A*D− (alph_off)*Bcom[1] − (f−1)*kx*Bcom[1]*D +2*k_x*Bcom[2] −(beta_in)*Bcom[1] + (gmma_out)*Ccom[1] d/dt (Bcom[2..(f−1)]) =(f−i+1)*kx*Bcom[i−1]*D − i*k_x*Bcom[i] − (f−i)*kx*Bcom[i]*D +(i+1)*k_x*Bcom[i+1] − (beta_in)*Bcom[i] + (gmma_out)*Ccom[i] d/dt(Bcom[f]) = kx*Bcom[i−1]*D − f*k_x*Bcom[i] − (beta_in)*Bcom[i] +(gmma_out)*Ccom[i] INIT Ccom[1..f] = 0 d/dt (Ccom[1..f]) =(beta_in)*Bcom[i] − (gmma_out)*Ccom[i] − (theta_c)*Ccom[i] INITA=Shh/(Kgli3*vff) INIT D=Do/(Kgli3*vff) INIT E=Eo/(Kgli3)Signal=1/(1+ce*D) ; fraction of unbound Smo, based on Scatchard rxnbetween Ptc and Smo kxfactor=12 ; acceleration of kon for multimeric Shhafter initial binding kx=alph_on*kxfactor k_x=alph_off ;*******************************************************************************;Define intracellular equations ; Nondimensional variables: ;G1 = (molGli1) / (L of intracellular liquid volume) / (Kgli3) ;G3 = (mol Gli3activator form) / (L of intracellular liquid volume) / (Kgli3) ;G3R =(mol Gli3R repressor form) / (L of intracellular liquid volume) /(Kgli3) ;*******************************************************************************d_G1=(alph)*Promoter + (beta)*Basal − G1 d/dt (G1) = (alph)*Promoter +(beta)*Basal − G1 d/dt (G3) = (gmma)/(G1+const) −G3*(1+(dlta)/(Kg3rc+Signal)) d/dt (G3R) = G3*(dlta)/(Kg3rc+Signal) − G3RG3o=5.81e−9 G3Ro=61.2e−9 G1o=1.63e−9 INIT G1=G1o/Kgli3 INIT G3=G3o/Kgli3INIT G3R=G3Ro/Kgli3 ;---------------------------------------------------------------------------------------------------;Define dimensional constants for intracellular equations ;---------------------------------------------------------------------------------------------------basfactor=130 kcatg=1.992732e−10 ; maximum rate of Gli synthesis(2.4e−10 M/min) rgbas=2.74e−10/basfactor ; Basal rate of Gli synthesis(vmax,g/100) kcatp=1.5e−10; maximum rate of Ptc synthesis (4.5e−10M/min) rbas=2.25e−9/basfactor ; Basal rate of Ptc synthesis (vmax,P/100)rg3b=3.1e−19 ; basal rate of gli3 synthesis (1.6e−19 M2/min)Kshh=8.3e−10 ; Dissociation constant for Shh-Ptc binding Kgli3=8.3e−10 ;used for Kptc kdeg=0.009 ; Degradation rate constant for Gli 1 (.009min−1) kdegp=0.09 ; Degradation rate constant for Ptc (.045-0.071 min−1)Kptc=3.32e−11 ; Half-maximal conc for Ptc which inhibits Smo signalingkg3r=0.0117 ; rate constant for the conversion of Gli3 to Gli3R (0.012min−1) Kg3rc=0.12 ; Sensitivity constant of the conversion to signalstrength bc=1 ; binding cooperativity tc=0.5 ; transcriptionalefficiency r=0.2 ; transcriptional repression afr=0.5 ; affinity ratioS=120 alph=kcatg/(Kgli3*kdeg) beta=rgbas/(Kgli3*kdeg)gmma=rg3b/(Kgli3*Kgli3*kdeg) dlta=kg3r/kdeg epsilon=kcatp/(kdeg*Kgli3)etta=rbas/(Kgli3*kdeg) const=1e−30

Results Chemical Conjugation

A recombinant rat Shh variant with a cysteine residue near theC-terminus was constructed, expressed, and purified via immobilizedmetal affinity chromatography. Conjugation of the recombinant proteinwas achieved on both pAAc and HyA with high efficiency. Using gelelectophoresis, it was apparent that the reaction produced a decrease inthe monomeric Shh band (FIG. 2) and the appearance of a high molecularweight conjugate. For pAAc (FIG. 2A) (MW=450,000), this produced a smearthrough the gel with an increasing mass as the Shh conjugation molarfeed ratio increased from 1:1 to 30:1. For HyA (MW=106 Da), the highmolecular weight conjugates did not penetrate deeply into the gel (FIG.2B). By contrast, simply mixing the Shh with raw pAAc or HyA did notalter the Shh mobility in the gel. Protein analysis of purified Shh:HyAreactions at 10:1 and 30:1 molar feed ratios performed in triplicateindicated that the reaction was reproducible with a high degree ofefficiency at approximately 70-75% (Table 2). Molar feed ratios of 1:1,5:1, 10:1, 20:1, and 30:1 produced Shh:HyA conjugates with molarsubstitution ratios of 0.6:1, 3.5:1, 7:1, 14:1, and 22:1, respectively.

TABLE 2 Determined conjugation ratios and coupling efficiencies forShh:HyA reactions at 30:1 and 10:1 molar feed ratios. Molar Ratio ofShh:HyA Percent Coupling Feed Ratio 30:1 10:1 30:1 10:1 Trial 1 21.806.91 73.3% 69.8% Trial 2 22.58 6.65 76.0% 67.1% Trial 3 22.09 7.02 74.3%70.8% Average 22.16 6.86 74.5% 69.2% Stdev 0.40 0.19 1.3% 1.9%

C3H10T1/2 Cell Bioactivity Assay

Through the use of the murine embryonic cell line C3H10T1/2, conjugationof the Shh was shown to dramatically alter the activity of the tetheredprotein when evaluated against actual Shh concentration in solution(FIG. 4). Only HyA-conjugated Shh could be tested using this cell line,as pAAc inhibited the differentiation that soluble Shh induces in thecell line. At low tethering (e.g., 3.5:1), the activity was decreased,with an estimated 10-fold increase in EC₅₀ of the Shh in solution,presumably due to steric hindrances that the large linear polymer causedwhen attached. The activity increased back to normal when theconjugation ratio reached 7:1. Beyond this, activity of the tethered Shhwas increased dramatically, with a 10-fold decrease in estimated EC₅₀values from the untethered Shh to the 22:1 construct.

CAM Angiogenesis Model

The CAM results indicated an increased potency for the conjugatedShh:HyA. Photographic analysis and quantification (FIGS. 5 and 6,respectively) revealed a statistically significant increase invasculature around the Shh-loaded samples compared to the negativecontrol within a close distance (0.1 mm) of the implant. While theShh:HyA conjugated at a 14:1 ratio had an increased average vesselnumber over both the negative control and unconjugated Shh at 0.1 cm, italso had a longer-range persistent increase over the negative control at0.25 cm. Although the soluble Shh also had an increased average vesselnumber over the negative control at this distance, this observation wasnot statistically significant.

Numerical Modeling of Multivalent Shh Bioactivity

Simple kinetic models of HyA:Shh conjugate binding, trafficking, anddownstream signal activation was developed. To focus on the effects ofconjugate multivalency, a number of assumptions were invoked in thesimplified models: Ptc receptor aggregation does not affect signaltransduction; ligand-internalization is not affected by valency;alkaline phosphatase activity is linearly proportional to Gli1 levels ina cell regardless of differentiation; and only two rates of conjugatebinding occur—an initial binding of the conjugate and a higher bindingrate for all additional Shh moieties from the conjugate to other Ptcreceptors (see Methods section for model equations and Table 1 forparameters). With these assumptions, two types of models were developed,one incorporating steric hindrance of HyA chains as a simple reductionin conjugate binding affinity to Ptc, and one neglecting any influenceof steric hindrance. These two models were termed “model with sterics”and “model without sterics,” respectively. Under these assumptions,modeling results indicated that increasing the conjugation ratio of Shhto its HyA carrier in the bioactivity assay should result in aprogressive increase in cell signaling and decrease in the EC₅₀ (FIG.7). The estimated EC₅₀ values from the experimental data werewell-matched using both types of kinetic models at the testedconjugation ratios and with the aforementioned assumptions (R²=0.7 forthe model without sterics; R²=0.8 for the model with sterics). For themodel without sterics, EC₅₀ values matched experimental results well athigh conjugation ratios, but the modeling results over estimated theEC₅₀ values for conjugation ratios 7:1 and lower (FIG. 7). Results fromthe model with sterics can correct for this deviation (FIG. 7).

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While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-13. (canceled)
 14. A treatment method comprising administering to anindividual in need thereof an effective amount of a pharmaceuticalcomposition comprising: a) a pharmaceutically acceptable excipient; andb) a conjugate of the formula:[X—(Y)Y_(n)]₁₀₋₅₀—Z, wherein X is a biologically active polypeptidehaving a molecular weight of from about 5 kDa to about 50 kDa; Y is anoptional linker moiety, wherein n is 0 or an integer from 1 to about 10;and Z is a biocompatible polymer having a molecular weight of at leastabout 450,000 Daltons, wherein the polypeptide is covalently linked tothe polymer directly or via the linker moiety.
 15. The method of claim14, wherein the polymer is a linear polymer comprising multiple subunitsselected from hyaluronic acid, acrylic acid, ethylene glycol,methacrylic acid, acrylamide, hydroxyethyl methacrylate, mannitol,maltose, glucose, arabinose, taurine, betaine, modified celluloses,hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl methyl cellulose, carboxymethylcellulose, modified starches, hydrophobically modified starch,hydroxyethyl starch, hydroxypropyl starch, amylose, amylopectin,oxidized starch, and copolymers thereof.
 16. The method of claim 14,wherein the biologically active polypeptide is a receptor, a ligand fora receptor, a growth factor, an angiogenic factor, a polypeptide thatinduces cell differentiation, an antibody, or a polypeptide thatinhibits cell proliferation.
 17. The method of claim 14, wherein thepolymer is linear poly(acrylic acid).
 18. The method of claim 14,wherein the polymer is hyaluronic acid.
 19. The method of claim 14,wherein the conjugate has the formula:[X—(Y)n]₁₀₋₂₅—Z.
 20. The method of claim 14, wherein the conjugate hasthe formula:[X—(Y)n]₂₅₋₅₀—Z
 21. The method of claim 14, wherein the biocompatiblepolymer has a molecular weight of at least 1×10⁶ Daltons.
 22. The methodof claim 14, wherein the biologically active polypeptide has a molecularweight of from about 10 kDa to about 50 kDa.
 23. The method of claim 14,wherein the biologically active polypeptide has a molecular weight offrom about 10 kDa to about 25 kDa.
 24. The method of claim 14, whereinthe biologically active polypeptide has a molecular weight of from about25 kDa to about 50 kDa.
 25. A treatment method comprising administeringto an individual in need thereof an effective amount of implantabledevice comprising a conjugate of the formula:[X—(Y)n]₁₀₋₅₀—Z, where X is a biologically active polypeptide having amolecular weight of from about 5 kDa to about 50 kDa; Y is an optionallinker moiety, wherein n is 0 or an integer from 1 to about 10; and Z isa biocompatible polymer having a molecular weight of at least about450,000 Daltons, wherein the polypeptide is covalently linked to thepolymer directly or via the linker moiety.
 26. A treatment methodcomprising administering to an individual in need thereof an effectiveamount of an implantable drug delivery device comprising a conjugate ofthe formula:[X—(Y)n]₁₀₋₅₀—Z, where X is a biologically active polypeptide having amolecular weight of from about 5 kDa to about 50 kDa; Y is an optionallinker moiety, wherein n is 0 or an integer from 1 to about 10; and Z isa biocompatible polymer having a molecular weight of at least about450,000 Daltons, wherein the polypeptide is covalently linked to thepolymer directly or via the linker moiety.